Detection and quantification of nucleic acid molecules constitutes a fundamental element in several diagnostic techniques. An essential feature of such techniques is the ability of a probe (a nucleic acid or nucleic acid analogue) to hybridize specifically to a complementary nucleic acid sequence. For hybridisation to occur some standard conditions have to be met regarding e.g. salt concentration and temperature, but the major determining factor is the number of fully matched nucleobases in the hybrid of two hybridizing strands. In hybrids of relatively short length, e.g. 6-10 basepairs, a single base pair mismatch will result in a drastic decrease in thermal stability, whereas the relative reduction of stability caused by a single base pair mismatch (or a deletion/insertion) becomes increasingly less with increasing length of the hybrid.
For diagnostic purposes, it is often desirable to identify a sequence of nucleobases which is present only in a particular gene or organism, but absent in any background nucleic acid that may be present in the sample. For a particular sequence of nucleobases to be statistically unique in a typical sample, like e.g. the human genome, the length of the sequence must be in the order of at least 18-20 bases, which on the other hand will enhance its capacity to accomodate mismatches, without dramatic loss of thermal stability.
While recognition of nucleic acids is generally possible by the use of duplex or triplex formation, today primarily duplex forming probes binding single stranded analyte nucleic acid via Watson-Crick (WC) basepairing, are used for diagnostic purposes. Although triplex structures, involving both WC and Hoogsteen binding (H), have been shown to be very thermically stable compared to duplexes, two conflicting properties have seemed to present an obstacle to their exploitation for diagnostic purposes: Because an uninterrupted strand of purine bases is normally required in a triplex forming target sequence, the chance of finding genus or species specific targets within a particular NA sequence is quite small since such a sequence would have to have a certain length in order to be specific. Even if longer, and thus, statistically unique purine sequences were present, the hybridizing properties of any complementary triplex forming probes (high binding capacity per basepair) would cause these to bind unspecifically also to all part sequences down to 6 or 7 bases in length, thus, loosing their specificity.
In WO 92/20702 it is disclosed that certain peptide nucleic acids can strand invade into double stranded DNA and bind DNA with high affinity. In WO 92/20703 the preparation and use of peptide nucleic acids (PNA) as diagnostic probes is disclosed. In WO 95/01370 it is disclosed that two molecules of identical homopyrimidine sequence can form stable triplex structures containing two molecules of the PNA and one molecule of single stranded DNA or RNA. These structures are disclosed for both therapeutic and diagnostic use.
In WO 96/02558 there are disclosed molecules containing two segments of identical sequence, but antiparallel orientation, linked by a bridging moiety. Such molecules are disclosed to exhibit a small but significant increase in Tm compared to the complex formed from independent PNAs each having the sequence of one of the segments. This is ascribed to the high local concentration of the covalently linked PNA strands. The segments disclosed are generally 6 or more bases in length. The linked peptide nucleic acids of this disclosure suffer from the priniciple problem that for sufficient sequence specificity of binding there is a need for a relatively large pyrimidine stretch, corresponding to a complementary purine stretch in the analyte nucleic acid, which would lead to very high levels of unspecific binding.
It is an object of the present invention to provide sequence specific complexes containing three independent strands bound by triple helix formation, wherein one of the strands is a short triplex forming probe molecule.